Genetic deficiency of adenosine deaminase (ADA), an enzyme of purine metabolism, is a cause of approximately 20% of cases of severe combined immunodeficiency (SCID). ADA-deficient SCID as been approached by gene transfer into T lymphocytes and hematopoietic stem cells (HSC), but no significant clinical benefit has been realized by the subjects. The availability of effective enzyme replacement therapy (PEGADA) for ADA-deficient SCID complicates the design and interpretation of clinical gene transfer research studies. In a clinical trial we began in 1993, CD34+ cells from the umbilical cord blood of three ADA-deficient SClD neonates were transduced with a retroviral vector carrying a normal human ADA cDNA and transplanted into the infants, who were also treated with PEG-ADA. The frequency of engrafted transduced HSC was low (-1t10, 000), but there was a selective increase of the frequency of gene-containing T lymphocytes (-1/10) as PEG-ADA was withdrawn.. There was not detectable expression from the retroviral vector in resting peripheral blood T lymphocytes, but expression could be induced to readily detectable levels upon in vitro stimulation of the T lymphocytes. Possibly, there was expression by the vector in activated thymocytes, conferring selective survival of gene-containing T lymphocytes during thymopoiesis. There was not a parallel selective increase of the frequency of gene-containing B lymphocytes and NK cells, and the absolute numbers of B lymphocytes and NK cells fell when PEG-ADA was stopped for two months. A recent study of ADA gene transfer to bone marrow HSC in which subjects were given modest dosages of cytoreductive chemotherapy and in which PEGADA enzyme therapy was withheld, has shown initial evidence for immune reconstitution, although whether this is attributable to the chemotherapy, the absence of PEG-ADA or both is unclear. Thus, a number of questions regarding the optimal approach to gene therapy for ADA-deficient SCID remain. A murine model of ADA-deficient SCID which reflects many major aspects of the human immunodeficiency disease has been developed by ADA gene knock-out, rescue of perinatal mortality by placental expression of an ADA transgene, and prolongation of post-natal survival by administration of PEG-ADA. We will use this model to study aspects of gene transfer to guide the development of future clinical trials of gene transfer research.